A partially defined medium, 294 devised for axenic cultivation of Entamoeba histolytica facilitates study of nucleic acid metabolism and furthers investigation of nutritional requirements of the parasite. An improved medium, TYSGM-9 has been devised for xenic cultivation of lumen dwelling protozoa inhabiting the alimentary tract of man. The bile factor stimulating growth of Giardia lamblia in vitro has been shown to be a low molecular weight compound. Development of a new cryopreservent permits freeze-preservation by controlled rate and uncontrolled slow freezing of 14 species of lumen dwelling protozoa (Entamoeba, Dientamoeba, Giardia, Trichomonas, and Crithidia), Medium 114, a partialy defined medium in which the carbohydrate content can be controlled precisely, has been used to study encystment of the laboratory model Entamoeba invadens under axenic conditions, and to facilitate study of the effects of specific bacterial extracts and metabolites on encystment of axenized E. histolytica.